Lipase mutant, immobilized lipase and application thereof in preparation of vitamin A palmitate

Abstract

The invention discloses a lipase mutant, immobilized lipase and application of the lipase mutant in preparation of vitamin A palmitate. Compared with SEQ ID NO: 1, the amino acid sequence of the lipase mutant has the difference of one or more amino acid residues selected from G55V, S68A, S70A, P114T and D171P. Compared with wild type lipase Lol-WT, the lipase mutant disclosed by the invention has the advantages that the enzyme activity is obviously improved, wherein the enzyme activity of the lipase mutant LolM6-1362 is most obviously improved. The immobilized enzyme (such as LolM6-1362-LX1080) prepared by utilizing the lipase mutant disclosed by the invention is simple in immobilization scheme, can be repeatedly applied for more than 30 times, is mild in reaction condition and high in reaction speed, and has an industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of enzyme catalysis technology, specifically, it relates to a lipase mutant, an immobilized lipase and its application in the preparation of vitamin A palmitate. Background Technology

[0002] Vitamin A is an essential trace element for the human body, possessing functions such as maintaining visual function, promoting bone growth and development, anti-inflammation, anti-oxidation, immune regulation, and anti-aging. Currently, vitamin A is widely used in pharmaceuticals, food, animal feed, and cosmetics.

[0003] Because vitamin A has an unsaturated double bond structure, it is easily damaged by high temperatures, oxides, acidic environments, or ultraviolet radiation, forming numerous byproducts. Therefore, commercially available vitamin A often exists in more stable ester forms, primarily vitamin A acetate and vitamin A palmitate. Compared to vitamin A acetate, vitamin A palmitate has better stability and pharmacological functions, making it suitable for use in food and pharmaceuticals, while vitamin A acetate is mainly used in animal feed. Furthermore, vitamin A palmitate commands a higher market price. Therefore, developing a synthetic process for vitamin A palmitate has promising market prospects.

[0004] Currently, the main production process for vitamin A palmitate uses the acyl chloride method, which generates a significant amount of toxic substances and byproducts, affecting the safety of the final product. Furthermore, the chemical synthesis of vitamin A palmitate requires high temperatures, which can damage the vitamin's structure and greatly increase equipment operating costs. In contrast, the enzyme-catalyzed synthesis of vitamin A palmitate offers advantages such as mild reaction conditions, high safety, and being environmentally friendly and pollution-free.

[0005] In China, most processes for preparing vitamin A palmitate using bio-enzymatic catalysis employ transesterification. Transesterification often requires a long conversion time to advance vitamin A acetate to complete the reaction, resulting in a low conversion rate. Furthermore, vitamin A palmitate and vitamin A acetate coexist after the reaction, making subsequent separation and extraction difficult.

[0006] Chinese patent CN112921064A discloses a process for synthesizing vitamin A palmitate by first preparing vitamin A alcohol through alcoholysis of vitamin A acetate, and then using an immobilized lipase from *Thermophilus combustor*. This process can advance the reaction to a relatively complete stage in a shorter time, but the amount of immobilized enzyme used is still relatively high and the number of repetitions is still relatively small. Therefore, developing lipases with excellent catalytic performance and a preparation process for vitamin A palmitate is expected to reduce the cost of enzyme use and improve the market competitiveness of vitamin A palmitate. Summary of the Invention

[0007] To address the limitations of existing immobilized lipases used in the preparation of vitamin A palmitate. 435 or To address the issue of the high cost of TL IM, this invention provides a lipase mutant, an immobilized lipase, and its application in the preparation of vitamin A palmitate. Specifically, this invention involves site-directed mutagenesis and screening of a lipase derived from *Lichtheimiaornata*, identifying a lipase mutant that still exhibits high conversion capacity against high concentrations of vitamin A. Further screening for suitable resins allows for the preparation of an immobilized enzyme, enabling highly efficient catalysis of the reaction between vitamin A and palmitic acid to produce vitamin A palmitate, demonstrating promising prospects for industrial application.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0009] The first aspect of the present invention provides a lipase mutant, wherein the amino acid sequence of the lipase mutant differs from the amino acid sequence shown in SEQ ID NO:1 by one or more amino acid residues selected from G55V, S68A, S70A, P114T, and D171P.

[0010] In some implementations, the differences are selected from any one of the following groups:

[0011] (1) G55V;

[0012] (2)S68A;

[0013] (3) S70A;

[0014] (4)P114T;

[0015] (5)D171P;

[0016] (6) G55V, S68A, S70A, P114T, D171P (corresponding to SEQ ID NO:3).

[0017] In some preferred embodiments, the amino acid sequence of the lipase mutant is shown in SEQ ID NO:3.

[0018] A second aspect of the present invention provides an isolated polynucleotide that encodes a lipase mutant as described in the first aspect of the present invention.

[0019] In some preferred embodiments, the nucleotide sequence of the isolated polynucleotide is shown in SEQ ID NO:4.

[0020] A third aspect of the present invention provides a recombinant expression vector comprising isolated polynucleotides as described in the second aspect of the present invention.

[0021] In some preferred embodiments, the backbone of the recombinant expression vector is a plasmid, granule, or viral vector; the viral vector is preferably a bacteriophage, retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector; the plasmid is preferably pUC, pET, pACYCDuet, or pETDuet, more preferably pET21a, pET24a, or pET28a.

[0022] The fourth aspect of the present invention provides a genetically engineered bacterium that expresses a lipase mutant as described in the first aspect of the present invention, or contains isolated polynucleotides as described in the second aspect of the present invention, or contains a recombinant expression vector as described in the third aspect of the present invention.

[0023] In some preferred embodiments, the originating organism of the genetically engineered bacteria is a fungus or a bacterium.

[0024] In some preferred embodiments, the starting bacteria are selected from any one of Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, and Escherichia coli.

[0025] In some further preferred embodiments, the originating bacterium is Escherichia coli, for example, Escherichia coli BL21(DE3).

[0026] The fifth aspect of the present invention provides a method for preparing a lipase mutant, the method comprising culturing genetically engineered bacteria as described in the fourth aspect of the present invention, and obtaining the lipase mutant from the culture.

[0027] In some preferred embodiments, the culture is a seed culture and / or an induction culture.

[0028] In some preferred embodiments, the seed culture refers to: culturing the genetically engineered bacteria at 30-40°C, for example, 37°C, with shaking for 12-20 hours, for example, 16 hours.

[0029] In some preferred embodiments, the induction culture includes: culturing the genetically engineered bacteria at 30-40°C, for example, 37°C, with shaking until OD... 600 When the concentration is 0.6-0.8, add IPTG and cool to 20-30℃, for example, 25℃, and incubate for 22-26 hours, for example, 24 hours.

[0030] In some preferred embodiments, the culture medium used for the seed culture is LB medium.

[0031] In some preferred embodiments, the culture medium used for the induction culture is TB medium.

[0032] In some preferred embodiments, during the induction culture, the genetically engineered bacteria are inoculated into the culture medium at an inoculation ratio of 0.5%-1.5% (v / v), preferably 1% (v / v).

[0033] In some preferred embodiments, the shaking culture is performed at a rotation speed of 150-300 rpm, for example, 200 rpm.

[0034] In some implementations, obtaining lipase mutants from cultures refers to collecting cell cultures, disrupting the cells, and purifying the proteins from the cell lysates to obtain the desired lipase mutants.

[0035] In some preferred embodiments, the protein separation and purification refers to ammonium sulfate precipitation.

[0036] A sixth aspect of the present invention provides an immobilized lipase comprising a resin and a lipase mutant as described in the first aspect of the present invention.

[0037] In some embodiments, the resin is a macroporous adsorption resin.

[0038] In some preferred embodiments, the macroporous adsorption resin comprises LX201A, LX201B or LX1080 of Xi'an Lanxiao Technology New Material Co., Ltd., for example LX1080.

[0039] In some further preferred embodiments, the amino acid sequence of the lipase mutant is shown in SEQ ID NO:3.

[0040] The seventh aspect of the present invention provides a method for preparing an immobilized lipase, the method comprising: contacting a lipase mutant as described in the first aspect of the present invention with a resin to prepare the immobilized lipase.

[0041] In some preferred embodiments, the resin is a macroporous adsorption resin.

[0042] In some preferred embodiments, the lipase mutant is used in the form of a free enzyme, a liquid enzyme, a crude enzyme solution, or cell fragments.

[0043] In this invention, free enzymes refer to enzyme molecules that exist in a soluble state in solution after being isolated and extracted from cells or tissues; liquid enzymes refer to liquid preparations formed by dissolving enzyme preparations in buffer or protective agents; crude enzyme solutions refer to enzyme-containing mixtures obtained through preliminary extraction such as tissue grinding and cell lysis; cell fragments refer to enzyme-containing mixtures obtained after cell membranes are destroyed by physical (ultrasound, high-pressure homogenization), chemical (lysis agent), or biological (enzymatic hydrolysis) methods.

[0044] In some preferred embodiments, the macroporous adsorption resin comprises LX201A, LX201B or LX1080 of Xi'an Lanxiao Technology New Material Co., Ltd., for example LX1080.

[0045] In some further preferred embodiments, the amino acid sequence of the lipase mutant is shown in SEQ ID NO:3.

[0046] The eighth aspect of the present invention provides a method for preparing vitamin A palmitate, the method comprising contacting and reacting a lipase mutant as described in the first aspect of the present invention or an immobilized lipase as described in the sixth aspect of the present invention with a substrate in a solvent to produce vitamin A palmitate; the substrate comprising retinol and palmitic acid.

[0047] In some embodiments, the solvent is n-hexane or n-heptane.

[0048] In some embodiments, the molar ratio of retinol to palmitic acid is 1:1.0 to 1.5, preferably 1:1.1 to 1.5.

[0049] In some embodiments, the mass ratio of the immobilized lipase to the palmitic acid is 1 to 5:35; preferably 3 to 5:35.

[0050] In some embodiments, the reaction temperature is 30–40°C; preferably 35°C.

[0051] In some embodiments, the reaction speed is 200-400 rpm; preferably 300 rpm.

[0052] In some embodiments, the reaction time is 0.5 to 1.5 hours; preferably 1 hour.

[0053] In some embodiments, the retinol is obtained by saponification of retinyl acetate with an alkali in an alcoholic solution.

[0054] In some embodiments, the base is KOH or NaOH, preferably KOH.

[0055] In some embodiments, the alcohol solution is methanol or ethanol, preferably methanol.

[0056] In some embodiments, the vitamin A acetate and the OH in the base - The molar ratio is 1:2 to 3, for example, 1:2.

[0057] In some embodiments, the molar ratio of the vitamin A acetate to the palmitic acid is 1:1.0 to 1.5, or 1:1.1 to 1.5.

[0058] In some embodiments, the saponification reaction is carried out at a temperature of 10–20°C, for example, 15°C.

[0059] In some embodiments, the saponification reaction takes 30 to 50 minutes, for example, 40 minutes.

[0060] A ninth aspect of the present invention provides a composition comprising vitamin A acetate, palmitic acid, a base, a lipase mutant as described in the first aspect of the present invention, or an immobilized lipase as described in the sixth aspect of the present invention; or, the composition comprises retinol, palmitic acid, a lipase mutant as described in the first aspect of the present invention, or an immobilized lipase as described in the sixth aspect of the present invention.

[0061] In some embodiments, (1) when the composition comprises vitamin A acetate, palmitic acid, a base, the lipase mutant, or the immobilized lipase:

[0062] The alkali is KOH or NaOH, preferably KOH;

[0063] And / or, the vitamin A acetate and the OH in the base - The molar ratio is 1:2 to 3, for example, 1:2;

[0064] And / or, the molar ratio of the vitamin A acetate to the palmitic acid is 1:1.0 to 1.5, for example, 1:1.1 to 1.5.

[0065] In some embodiments, (2) when the composition comprises retinol, palmitic acid, the lipase mutant, or the immobilized lipase:

[0066] The molar ratio of vitamin A alcohol to palmitic acid is 1:1.0 to 1.5, preferably 1:1.1 to 1.5;

[0067] And / or, the mass ratio of the immobilized lipase to the palmitic acid is 1 to 5:35; preferably 3 to 5:35.

[0068] The tenth aspect of this invention provides a reaction end product system for the catalytic synthesis of vitamin A palmitate using retinol as a substrate, the reaction end product system comprising:

[0069] Vitamin A palmitate;

[0070] Vitamin A (retinol);

[0071] Palmitic acid; and

[0072] Lipase mutants as described in the first aspect of the present invention or immobilized lipases as described in the sixth aspect of the present invention.

[0073] In some embodiments, the retinol content in the final product system is less than 1.1%.

[0074] In some embodiments, the retinol content in the final product system is less than 0.2%.

[0075] The eleventh aspect of the present invention provides the use of a lipase mutant as described in the first aspect of the present invention, an isolated polynucleotide as described in the second aspect of the present invention, a recombinant expression vector as described in the third aspect of the present invention, a genetically engineered bacterium as described in the fourth aspect of the present invention, an immobilized lipase as described in the sixth aspect of the present invention, or a composition as described in the ninth aspect of the present invention in the preparation of vitamin A palmitate.

[0076] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0077] The reagents and raw materials used in this invention are all commercially available.

[0078] The positive and progressive effects of this invention are as follows:

[0079] The lipase mutants obtained by this invention show significantly improved enzyme activity compared to the wild-type lipase Lol-WT, with the most significant increase observed in the lipase mutant LolM6-1362. The immobilization scheme for the immobilized lipase (e.g., LolM6-1362-LX1080) prepared using the lipase mutants of this invention is simple, can be repeated more than 30 times, requires less immobilized enzyme, has milder reaction conditions, and a faster reaction rate. Detailed Implementation

[0080] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0081] The lipase mutant constructed in this invention is a mutant of the wild-type lipase Lol-WT (its amino acid sequence is shown in SEQ ID NO:1) derived from *Lichtheimia ornata*. It is a novel protein formed by one or more substitutions (G55V, S68A, S70A, P114T, D171P) in five amino acids of the sequence in SEQ ID NO:1. The GenBank accession number for SEQ ID NO:1 is XP_058345984.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:2, which has been optimized based on the codon bias of *Escherichia coli*.

[0082] To obtain lipases with higher enzyme activity, this invention rationally designed point mutations to the gene sequence SEQ ID NO:2 of SEQ ID NO:1, resulting in mutants with significantly enhanced lipase activity, including one or more combinations of the following point mutations: glycine at position 55 replaced by valine (G55V), serine at position 68 replaced by alanine (S68A), serine at position 70 replaced by alanine (S70A), proline at position 114 mutated by threonine (P114T), and lipid at position 171 mutated by proline (D171P). In particular, the mutant with the amino acid sequence shown in SEQ ID NO:3 exhibits significantly higher lipase activity compared to the wild-type enzyme SEQ ID NO:1.

[0083] The coding genes, expression cassettes and plasmids containing these genes, and transformants containing the plasmids involved in this invention can all be obtained through genetic engineering construction methods well known to those skilled in the art.

[0084] The transformant can be any microorganism suitable for expressing lipase mutants, including bacteria and fungi. Preferred microorganisms are Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, with Escherichia coli being preferred, and Escherichia coli BL21(DE3) being more preferred.

[0085] When preparing the lipase of the present invention into an immobilized enzyme using immobilization techniques known in the art, the lipase used may be an unpurified crude enzyme solution or a partially or completely purified enzyme.

[0086] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, all percentage contents mentioned are mass percentage contents.

[0087] The molecular biology experiments involved in the examples include plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, culture medium preparation, etc., and were conducted in accordance with "Molecular Cloning: A Laboratory Manual" (3rd Edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002.

[0088] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2 (LB solid medium with an additional 20 g / L agar powder).

[0089] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4·3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5.

[0090] HPLC determination conditions for the substrates vitamin A acetate, retinol, and the product vitamin A palmitate:

[0091] Chromatographic column: Shimadzu GL Inertsil ODS-3 liquid chromatography column, 5μm, 4.6×250mm;

[0092] Mobile phase: methanol.

[0093] HPLC detection conditions: detection time: 50 min; detection wavelength: 326 nm; column oven temperature: 35 ℃; pump speed: 1.0 ml / min; injection volume: 10 μl.

[0094] The molar conversion rate of vitamin A palmitate (%) = the molar amount of vitamin A palmitate converted to vitamin A / the molar amount of vitamin A acetate added at the beginning of the reaction × 100%.

[0095] Example 1: Construction of Recombinant Escherichia coli with Wild-Type Lipase Gene

[0096] The lipase Lol-WT from Lichtheimia ornata was synthesized by Suzhou Genewise Biotechnology Co., Ltd. The gene was optimized according to the codon preference of Escherichia coli, and the optimized gene sequence is SEQ ID NO:2. After the gene was synthesized, it was cloned into the NdeI and HindIII restriction sites of the vector pET28a (purchased from BioWind) to obtain the recombinant plasmid pET28a-Lol-WT.

[0097] The recombinant plasmid pET28a-Lol-WT was transformed into the expression host Escherichia coli BL21(DE3) (purchased from Hangzhou Baosai Biotechnology Co., Ltd.) by chemical transformation to obtain recombinant Escherichia coli Lol-WT that can express wild-type lipase.

[0098] Example 2: Constructing a Mutation Point Library through Semi-Rational Design

[0099] The substrate binding sites in the Lol protein pocket of the lipase were predicted using Discovery Studio software. Using pET28a-Lol-WT as the starter plasmid, single-point saturation mutagenesis and iterative saturation mutagenesis were performed at positions G55, S68, S70, P114, and D171. The mutant proteins were screened by transforming recombinant plasmids containing point-mutated Lol proteins (pET28a as the vector) into *E. coli* BL21(DE3) competent cells to obtain mutant transformants. Recombinant *E. coli* Lol-WT expressing wild-type lipase was used as a control. Seed cultures were obtained by inoculating each culture in 5 mL of LB medium (containing 50 μg / mL kanamycin) at 37°C with shaking at 200 rpm for 16 hours. Each seed culture was then inoculated at a 1% ratio into fresh TB medium (containing 50 μg / mL kanamycin) and cultured at 37°C with shaking at 200 rpm until OD (digestive activity) was reached. 600 Add 0.6-0.8 μL of IPTG to a final concentration of 0.1 mM, cool to 25°C and induce culture for 24 hours. Centrifuge at 10,000 rpm for 5 min at 4°C and collect the bacterial cells. Add 50 mM Tris-HCl pH 8.0 buffer at a weight ratio of 1:3 to the collected wet bacterial cells, homogenize using a high-pressure homogenizer, centrifuge the homogenate to discard the precipitate, and the supernatant is the corresponding enzyme.

[0100] Example 3: Screening of lipase mutants

[0101] Using p-nitrophenyl butyrate (pNPB) as a substrate, the enzyme activity of each lipase mutant obtained in Example 2 was detected. The unit lipase activity (U) was defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol (pNP) from pNPB per minute.

[0102] Detection method: The reaction system (3 mL) contains: 50 mM Tris-HCl buffer (pH 8.0), 4% (v / v) anhydrous ethanol and 0.5 mM pNPB substrate (pNPB is prepared into a 50 mM stock solution with acetonitrile). The reaction temperature is 25℃. Add 0.2 mL of preheated enzyme solution (enzyme solution diluted appropriately). The reaction time is 5 min. The detection wavelength is 405 nm.

[0103] Table 1 Screening of lipase mutants

[0104] Wild type / mutant number Wild-type / mutant mutation point relative enzyme activity % Lol-WT No mutation 100 LolM1-1254 G55V 211 LolM2-387 S68A 530 LolM3-53 S70A 161 LolM4-298 P114T 205 LolM5-977 D171P 278 LolM6-1362 G55V / S68A / S70A / P114T / D171P 763

[0105] As shown in the table, mutants LolM1-1254, LolM2-387, LolM3-53, LolM4-298, LolM5-977, and LolM6-1362 with significantly increased enzyme activity were obtained. Among them, mutant LolM6-1362 showed the most significant increase in enzyme activity. Its amino acid sequence is shown in SEQ ID NO:3, and it contains five point mutations: G55V, S68A, S70A, P114T, and D171P. Its nucleotide sequence is shown in SEQ ID NO:4. Compared with the wild-type enzyme SEQ ID NO:1, the enzyme activity of mutant protein LolM6-1362 was increased by 6.63 times.

[0106] Example 4: Preparation of Immobilized Lipase

[0107] (I) Preparation of immobilized lipase LolM6-1362-LX1080

[0108] (1) Preparation of crude enzyme solution

[0109] After the induction expression was completed, the culture medium was centrifuged at 12000 rpm for 5 minutes, and 500 g of bacterial sludge was collected. This sludge was then added to 1500 mL of 50 mM Tris-HCl (pH 8.0) buffer, mixed thoroughly, and homogenized using a homogenizer. The supernatant was collected by centrifugation, and 125 mL of 10% PEI flocculant (purchased from Adamas-life) was slowly added. After thorough mixing, the supernatant was collected by centrifugation, and 1500 mL of saturated ammonium sulfate solution was added. After thorough mixing, the supernatant was discarded by centrifugation, and the precipitate was reconstituted in 600 mL of 50 mM Tris-HCl pH 8.0 buffer to obtain the crude enzyme solution. The enzyme activity of the crude enzyme solution was determined to be 112.8 U / mL using pNPB as a substrate.

[0110] (2) Pretreatment of macroporous adsorption resin

[0111] The macroporous adsorption resin LX1080 (purchased from Xi'an Lanxiao Technology New Material Co., Ltd.) to be treated was soaked in deionized water for 3 hours and then filtered to remove water. It was then soaked in anhydrous ethanol for 12 hours and then filtered to collect the resin. The resin was repeatedly washed with deionized water to remove residual ethanol and then soaked in deionized water for later use. Before use, it was filtered to remove moisture.

[0112] (3) Immobilization of lipase LolM6-1362

[0113] Accurately weigh 100 g (wet weight) of pretreated macroporous adsorption resin LX1080, add 400 mL of crude enzyme solution, place on a rotary mixer, and adsorb at 4℃ and 80 rpm for 24 h. After the reaction, wash three times repeatedly with 50 mM Tris-HCl buffer, collect the carrier and air dry at room temperature to obtain immobilized lipase LolM6-1362-LX1080. The enzyme activity of immobilized lipase LolM6-1362-LX1080 was determined to be 523.3 U / g using pNPB as a substrate.

[0114] (II) Preparation of immobilized lipase LolM6-1362-LX201A

[0115] The immobilized enzyme was prepared using the same method as LolM6-1362-LX1080, except that the resin used was macroporous adsorption resin LX201A. The enzyme activity of the immobilized lipase LolM6-1362-LX201A was determined to be 320.1 U / g using pNPB as a substrate.

[0116] (III) Preparation of immobilized lipase LolM6-1362-LX201B

[0117] The immobilized enzyme was prepared using the same method as LolM6-1362-LX1080, except that the resin used was macroporous adsorption resin LX201B. The enzyme activity of the immobilized lipase LolM6-1362-LX201B was determined to be 265.0 U / g using pNPB as a substrate.

[0118] Example 5: Synthesis of Vitamin A Palmitate

[0119] (1) Preparation of Vitamin A alcohol

[0120] Accurately weigh 30 g of vitamin A acetate (0.09 mol) and 75 mL of methanol into a three-necked flask. Replace the air in the flask with nitrogen. While stirring at 100 rpm, slowly add 40 mL of 260 g / L potassium hydroxide methanol solution. Perform alcoholysis at 15 °C for 40 min. Add 80 mL of water to the alcoholysis solution, and extract the reaction mixture twice with 200 mL of n-heptane. Separate and combine the n-heptane phases, and wash three times with water. HPLC analysis of the n-heptane extract showed an alcoholysis conversion rate of 99.9%.

[0121] (2) Esterification of Vitamin A

[0122] Take all the vitamin A retinol solution synthesized in step (1) (containing 0.09 mol vitamin A retinol) and 35 g of palmitic acid into a three-necked flask, and add 5 g of the immobilized enzymes (LolM6-1362-LX1080, LolM6-1362-LX201A and LolM6-1362-LX201B) prepared in Example 4 respectively. React at 35℃ and 300 rpm under reduced pressure for 1 h. The molar conversion rates of vitamin A palmitate in the reaction solution were 99.7%, 63.2%, and 51.0% respectively, as determined by HPLC.

[0123] Example 6: Ratio of palmitic acid to vitamin A acetate

[0124] Vitamin A palmitate was synthesized using palmitic acid to vitamin A acetate at molar ratios of 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, and 1.0:1, respectively. The effect of this ratio on the conversion rate was investigated. The operating procedures and reaction conditions were the same as in Example 5. HPLC analysis showed that the molar conversion rates of vitamin A palmitate were 99.8%, 99.8%, 99.7%, 99.7%, 99.6%, and 98.9%, respectively. When the molar ratio of palmitic acid to vitamin A acetate was 1.1:1, the molar conversion rate of vitamin A palmitate reached 99.6%.

[0125] Example 7: Dosage of Immobilized Lipase

[0126] Vitamin A palmitate was synthesized using immobilized lipase prepared in Example 4 at concentrations of 5g, 4g, 3g, 2g, and 1g, respectively. The effect of this immobilized lipase on the conversion rate was investigated. The operating procedures and reaction conditions were the same as in Example 5. HPLC analysis showed that the molar conversion rates of vitamin A palmitate were 99.7%, 99.7%, 99.7%, 99.6%, and 99.6%, respectively. When the mass ratio of immobilized lipase to vitamin A acetate was 1:30 (g / g), the molar conversion rate of vitamin A palmitate reached 99.6%, demonstrating that the immobilized enzyme described in this invention has high catalytic efficiency, requires less enzyme during the reaction, and saves on the production cost of vitamin A palmitate.

[0127] Example 8: Number of times the immobilized enzyme was reused

[0128] After the reaction was completed, the immobilized enzyme was recovered, washed, and used in the next reaction to investigate the effect of the number of times it was reused on the conversion rate. The operation steps and reaction conditions were the same as in Example 5. Vitamin A palmitate was synthesized using the immobilized enzyme reused 1, 10, 20, 30, 40, and 50 times, respectively. HPLC analysis showed that the molar conversion rates of vitamin A palmitate were 99.7%, 99.5%, 99.3%, 99.0%, 98.1%, and 95.5%, respectively. Even after 30 reuses, the molar conversion rate of vitamin A palmitate using the immobilized enzyme described in this invention still reached 99.0%, demonstrating that the immobilized enzyme described in this invention has good stability and maintains high enzyme activity after repeated recovery, which is beneficial for saving the production cost of vitamin A palmitate.

[0129] The sequence used in this invention is as follows:

[0130] SEQ ID NO:1: Wild-type lipase Lol amino acid sequence from *Lichtheimia ornata*

[0131] MRFYSVVSLLVVSICTYGVSGVPVQIDARDKSYVPEQYPLKMNGPLPEGVSVIQGYCENCTMYPEENSVSALSSSKQDYRTASESEIKTHTFYTALSANAYCRNVIPGGRWSCPHCDVTSNLEITKTFSTLITDTNVMVAVGKKEKTIYIAFRGTNSIRNAIADIVFV PVDYPPVDGAKVHKGFLDSYNEVQDQLVAEVKKQLDNHPGYKIVVTGHSLGGATAVLCALDLYHHGHHNIEIYTQGQPRVGTPAFAKYVIGTKIPYQRLVNERDIVPHLPPGAFGFLHAGEEFWIMKDSSLRVCPNGIETDDCSNSIVPFTSVIDHLSYLDMNTGLCL

[0132] SEQ ID NO:2: Wild-type lipase Lol from *Lichtheimia ornata*, optimized based on *Escherichia coli* codon bias.

[0133]

[0134] SEQ ID NO:3: Amino acid sequence of mutant lipase LolM6-1362 (bold amino acids are those that differ from the wild type)

[0135] MRFYSVVSLLVVSICTYGVSGVPVQIDARDKSYVPEQYPLKMNGPLPEGVSVIQVYCENCTMYPEENAVAALSSSKQDYRTASESEIKTHTFYTALSANAYCRNVIPGGRWSCTHCDVTSNLEITKTFSTLITDTNVMVAVGKKEKTIYIAFRGTNSIRNAIADIVFV PVPYPPVDGAKVHKGFLDSYNEVQDQLVAEVKKQLDNHPGYKIVVTGHSLGGATAVLCALDLYHHGHHNIEIYTQGQPRVGTPAFAKYVIGTKIPYQRLVNERDIVPHLPPGAFGFLHAGEEFWIMKDSSLRVCPNGIETDDCSNSIVPFTSVIDHLSYLDMNTGLCL

[0136] SEQ ID NO:4: Nucleic acid sequence encoding mutant lipase LolM6-1362

[0137]

[0138] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A lipase mutant, characterized in that, The amino acid sequence of the lipase mutant has one or more amino acid residues different from the sequence shown in SEQ ID NO: 1, which are selected from G55V, S68A, S70A, P114T and D171P.

2. The lipase mutant according to claim 1, wherein The difference is selected from any one of the following groups: (1) G55V; (2) S68A; (3) S70A; (4) P114T; (5) D171P; (6) G55V, S68A, S70A, P114T and D171P; Preferably, the amino acid sequence of the lipase mutant is shown in SEQ ID NO:

3.

3. An isolated polynucleotide, comprising, The isolated polynucleotide encodes the lipase mutant as claimed in claim 1 or 2; Preferably, the nucleotide sequence of the isolated polynucleotide is shown in SEQ ID NO:

4.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated polynucleotide as claimed in claim 3; Preferably, the backbone of the recombinant expression vector is a plasmid, a cosmid or a viral vector; the viral vector is preferably a bacteriophage, a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector; the plasmid is preferably pUC, pET, pACYCDuet or pETDuet, more preferably pET21a, pET24a or pET28a.

5. A genetically engineered bacterium, characterized by, The genetically engineered bacteria express the lipase mutant as claimed in claim 1 or 2, or comprise the isolated polynucleotide as claimed in claim 3, or comprise the recombinant expression vector as claimed in claim 4; Preferably, the starting bacteria of the genetically engineered bacteria are fungi or bacteria; More preferably, the starting bacteria are selected from any one of Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae and Escherichia coli; Further preferably, the starting bacteria are Escherichia coli, for example Escherichia coli BL21 (DE3).

6. A method of preparing a lipase mutant, characterized in that, The method comprises culturing the genetically engineered bacteria as claimed in claim 5 to obtain the lipase mutant from the culture; Preferably, the culture is seed liquid culture and / or induction culture; The seed liquid culture preferably refers to culturing the genetically engineered bacteria at 30-40°C, for example 37°C, for 12-20h, for example 16h, with shaking. The induction culture preferably comprises: culturing the genetically engineered bacteria at 30-40℃, such as 37℃, with shaking to an OD 600 0.6-0.8, adding IPTG, and culturing at 20-30℃, such as 25℃, for 22-26h, such as 24h. More preferably, the medium used in the seed liquid culture is LB medium; and / or, the medium used in the induction culture is TB medium; and / or, in the induction culture, the genetically engineered bacteria are inoculated into the medium at an inoculation ratio of 0.5%-1.5% (v / v), preferably 1% (v / v); and / or, the shaking speed is 150-300rpm, for example 200rpm.

7. An immobilized lipase characterized in that, The immobilized lipase comprises a resin and the lipase mutant as claimed in claim 1 or 2; Preferably, the resin is a macroporous adsorption resin; More preferably, the macroporous adsorption resin comprises LX201A, LX201B or LX1080 of Xi'an Lanxiao Science and Technology New Material Co., Ltd., for example LX1080; Further preferably, the amino acid sequence of the lipase mutant is shown in SEQ ID NO:

3.

8. A method of preparing an immobilized lipase, characterized by, The method comprises: The lipase mutant as claimed in claim 1 or 2 is contacted with a resin to prepare the immobilized lipase; Preferably, the resin is a macroporous adsorption resin; and / or, the lipase mutant is in the form of free enzyme, liquid enzyme, crude enzyme liquid or cell broken product; More preferably, the macroporous adsorption resin comprises LX201A, LX201B or LX1080 of Xi'an Blue Sky Science and Technology New Material Co., Ltd., for example, LX1080; Further preferably, the amino acid sequence of the lipase mutant is shown in SEQ ID NO:

3.

9. A process for the preparation of vitamin A palmitate, characterized in that, The method comprises contacting and reacting the lipase mutant as claimed in claim 1 or 2 or the immobilized lipase as claimed in claim 7 with a substrate in a solvent to produce vitamin A palmitate; the substrate comprises vitamin A alcohol and palmitic acid; Preferably, the solvent is n-hexane or n-heptane; and / or, the molar ratio of the vitamin A alcohol to the palmitic acid is 1:1.0-1.5, preferably 1:1.1-1.5; and / or, the mass ratio of the immobilized lipase to the palmitic acid is 1-5:35; preferably 3-5:35; and / or, the temperature of the reaction is 30-40℃; preferably 35℃; and / or, the rotation speed of the reaction is 200-400rpm; preferably 300rpm; and / or, the time of the reaction is 0.5-1.5h; preferably 1h; More preferably, the vitamin A alcohol is obtained by saponification reaction of vitamin A acetate with a base in an alcohol solution; Further preferably, the base is KOH or NaOH, preferably KOH; and / or, the alcohol solution is methanol or ethanol, preferably methanol; and / or the OH of the vitamin A acetate and the OH of the base - are in a molar ratio of 1:2 to 3, for example 1:

2. and / or, the molar ratio of the vitamin A acetate to the palmitic acid is 1:1.0-1.5, for example, 1:1.1-1.5; and / or, the temperature of the saponification reaction is 10-20℃, for example, 15℃; and / or, the time of the saponification reaction is 30-50min, for example, 40min.

10. A composition characterized in that, The composition comprises vitamin A acetate, palmitic acid, a base, the lipase mutant as claimed in claim 1 or 2 or the immobilized lipase as claimed in claim 7; or, the composition comprises vitamin A alcohol, palmitic acid, the lipase mutant as claimed in claim 1 or 2 or the immobilized lipase as claimed in claim 7; Preferably, (1) when the composition comprises vitamin A acetate, palmitic acid, a base, the lipase mutant or the immobilized lipase: the base is KOH or NaOH, preferably KOH; and / or the OH of the vitamin A acetate and the OH of the base - are in a molar ratio of 1:2 to 3, for example 1:

2. and / or, the molar ratio of the vitamin A acetate to the palmitic acid is 1:1.0-1.5, for example, 1:1.1-1.5; (2) when the composition comprises vitamin A alcohol, palmitic acid, the lipase mutant or the immobilized lipase: the molar ratio of the vitamin A alcohol to the palmitic acid is 1:1.0-1.5, preferably 1:1.1-1.5; and / or, the mass ratio of the immobilized lipase to the palmitic acid is 1-5:35; preferably 3-5:

35.

11. A reaction end product system catalytically produced from vitamin A alcohol as a substrate to vitamin A palmitate, characterized by, The reaction end product system comprises: vitamin A palmitate; vitamin A alcohol; palmitic acid; and the lipase mutant of claim 1 or 2 or the immobilized lipase of claim 7; Preferably, the content of vitamin A alcohol in the final product system is less than 1.1%; More preferably, the content of vitamin A alcohol in the final product system is less than 0.2%.

12. Use of the lipase mutant of claim 1 or 2, the isolated polynucleotide of claim 3, the recombinant expression vector of claim 4, the genetically engineered bacterium of claim 5, the immobilized lipase of claim 7 or the composition of claim 10 in the preparation of vitamin A palmitate.